The RNA polymerases play a central role in gene expression. The isolation of the genes encoding yeast RNA polymerase II subunits provides us with a unique opportunity to systematically investigate the functions of this complex enzyme using a combination of genetics and biochemistry in Saccharomyces. These investigations should lay the foundation for ultimately understanding how RNA polymerase II is recruited to promoters and how the enzyme responds to signals to initiate, elongate and terminate transcription. The experiments described in this proposal are designed to help elucidate the functions of the RNA polymerase II subunits in various stages of transcription, to identify additional factors that are involved in each of these stages, and to deduce how RNA polymerase II and these accessory factors contribute to the control of gene expression. To accomplish these goals, the four specific aims of this proposal are: 1) to identify RNA polymerase II subunit mutations that affect transcription initiation, elongation and termination using in vivo and in vitro assays; 2) to use RNA polymerase II subunit mutations that affect transcription initiation, elongation and termination to identify genes encoding accessory factors by isolating and characterizing second-site suppressors; 3) to identify genes whose products affect CTD function during initiation by isolating and characterizing suppressors of CTD truncation mutations; and 4) to develop new biochemical assays that can reveal interactions between RNA polymerase II subunits and other proteins in vitro, especially those that are relatively weak or transient. The health relatedness of this project derives from its contribution to the understanding of basic molecular mechanisms that control gene expression.